Abstract. Hermaphroditic freshwater clams in the genus Corbicula produce non-reductional spermatozoa. The DNA content of spermatozoa was almost identical with that of somatic cells in C. leana from Mie Prefecture, Japan. Hermaphroditic C. aff. fluminea from Saga Prefecture and C. juminea from Taiwan also produce non-reductional spermatozoa. On the other hand, spermatozoa of the dioecious C. sandai had half the DNA found in somatic cells. Analysis of chromosome numbers suggests that C. leana (3n = 54 in somatic cells and 18 in meiotic cells) from Mie Prefecture and C. aff. Jluminea (2n = 36 in gills and 18 bivalents in meiotic cells) from Saga Prefecture are triploids and diploids, respectively. C. leana, C. aff. fluminea, and C. juminea may lack either first or second meiosis, resulting in non-reductional spermatozoa. We assume that gynogenetic reproduction occurs in both species; maternal chromosomes are also nonreductional, and spermatozoa activate development of the eggs, but do not contribute to the offspring.

(Okamoto and Arimoto, 1986). Okamoto and Arimoto (1986) suggested the possibility that C. leana reproduces by gynogenesis, which would account for the odd chromosome number: i.e., gynogenetic eggs are usually meiotically unreduced, and spermatozoa activate the development of eggs, but the paternal genome does not contribute to the offspring. Analysis of allozyme variation in C. Zeana also revealed that the species has much lower genetic variability than C. juponica and C. sandui (Sakai et al., 1994). In this study, we compare chromosome numbers and DNA content of somatic cells and spermatozoa in the hermaphroditic Corbicula species to test the gynogenesis hypothesis proposed by Okamoto and Arimoto (1986).

Materials

and Methods

The Corbicula species were identified according to Habe (1977). C. leana and C. aff. juminea were collected in Japan from an irrigation canal in Meiwa, Mie Prefecture, and from the Tade River, Saga Prefecture, respectively. In this study we use the name C. aff. jluminea for clams collected from Saga Prefecture because the morphology of the lateral teeth in these specimens was very similar to that of C. Jluminea (Komaru et al., 1998). C. jluminea was also obtained from a cultured population in Shin Wu, Taiwan. For comparison using microfluorometry, specimens of C. sandai from Lake Biwa, Shiga Prefecture, were obtained at the market in Ootsu city.

Introduction The freshwater clam Corbicula leana has a special mode of reproduction: it is hermaphroditic and broods its larvae in the inner demibranchs (Miyazaki, 1936; Ikematsu and Yamane, 1977). A recent study of the chromosomes of C. leana showed that it has 54 somatic chromosomes, which, judging from its karyotype, are triploids

Chromosomes Received 3 April 1997; accepted 19 August 1997. * To whom correspondence should be addressed. E-mail: bio.mie-u.ac.jp

The clams were treated with 0.002% colchicine for 45 h. Gills and gonads were dissected out with scissors,

treated with hypotonic solution (8 mM KCl), and fixed with Carnoy’s fixative according to the procedure of Okamoto and Arimoto (1986). Cells were isolated from either gill or gonads, placed on slides, and stained with 2% Giemsa in phosphate buffer (pH 7.2). At least 4 clams were examined for each case (i.e., each combination of species and tissue type). DNA microjborometry To compare the relative DNA content of spermatozoa and somatic tissue, cells were isolated on a glass slide by cutting a small piece of gonad or mantle in distilled water with a scalpel and air-drying it before fixing it with 70% ethanol. Spermatozoa and somatic cells from one individual were placed on the same slide. The cells were stained with the DNA-specific dye DAPI, and the relative DNA content (fluorescence intensity) per cell was estimated by microfluorometry as in Komaru et al. (1988). Spermatozoa could be easily distinguished from other sperm-

atogenic cells because of their elongate and curved morphology. Results

Mitotic

and meiotic chromosomes

In four specimens of C. leana, we counted 54 chromosomes in the mitotic metaphase plates of somatic tissue (Fig. 1A) and about 18 chromosomes in the gonad (Fig. 1B). In C. aff. Jluminea, we observed 36 chromosomes in the mitotic metaphase in 16 cells, but the haploid number of 18 in 8 gonadal cells. Pachytene (Fig. lD), diakinesis or metaphase I (Fig. 1E) figures were observed in the meiotic cells. Relative DNA content of spermatozoa and somatic cells In C. leana, C. aff.JEuminea, and C. JEuminea, the relative content of DNA in the spermatozoa was also identical

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Figure 2. Histograms of fluorescence intensity (relative DNA content) in spermatozoa and somatic cells of Corbicula leana from Mie Prefecture, C. aff. juminea from Saga, C. Jluminea from Taiwan, and C. sandai from Shiga. Each histogram was derived from one individual. DNA content per cell was measured as fluorescence intensity.

to that in the somatic cells (Fig. 2. and Table I). In C. sun&i, however, the DNA content of the spermatozoa was half that of the somatic cells. Discussion Okamoto and Arimoto (1986) reported chromosome numbers of 3n = 54 for C. leanu, 2n = 38 for C. japonica, Table Relative DNA content species uf Corbicula

Each mean value and s;tandard deviation (SD) was derived from one individual. The numbers in parentheses (n) are the number of cells measured.

53) 16) 26) 23) 44) 24) 48) 56) 26) 26) 34) 40) 38) 38) 38) 16)

and 2n = 36 for C. sandai. In the present study, the chromosome number of C. aff.$uminea was determined to be 2n = 36 in gill tissue and 18 bivalents in gonads, indicating that C.JEumineais a diploid species.In contrast, C. Zeanahas a somatic chromosome number of 54, but in the gonads, three homologous chromosomes form 18 trivalents. The synapsis of three homologous chromosomes should be incomplete. These results suggest that C. leana is a triploid species. The microfluorometric measurementsof DNA revealed that the hermaphroditic species C. leana and C. jhminea produced non-reductional spermatozoa; i.e., sperm and somatic cells had the same DNA content. On the other hand, the dioecious C. sandai produced reductional spermatozoa; the DNA content of sperm was half that of somatic cells. These results suggest that the cytokinesis of first or second meiosis in the spermatocytes of both C. leana and C. Jluminea is abortive, and only one equal division occurs. Consequently, meiosis during spermatogenesismay be non-reductional, resulting in spermatozoa with somatic ploidy levels. It is possible that triploid C. leana and diploid C. jluminea reproduce by gynogenesis, as O’Foighil and ThiriotQuievreux (1991) observed in the bivalve Lasaea. In gynogenetic development, spermatozoa stimulate the development of eggs but do not contribute paternal genome to the offspring. In this case the somatic chromosome number may be restored to the eggsby premeiotic endomitosis

NON-REDUCTIONAL

or abortive meiosis (Cuellar, 1987). How did the triploid C. leana evolve from a diploid ancestral species? We assume that the diploid gynogenetic C. jhminea arose first. Later, the triploid C. Zeana may have evolved from the diploid gynogen by fusion of diploid and haploid gametes. The processes of meiosis and fertilization of eggs should be observed in C. leana and C. jluminea to understand the reproductive processes in these species with non-reductional spermatozoa. Species of Corbicula show polymorphic shell color and morphology (Kuroda, 1938; Morton, 1986). The taxonomy of the Corbiculidae is in disarray as a result of intraspecific variation in shell morphology and insufficient biological information. Corbiculu taxonomy cannot be clarified on the basis of shell morphology alone; as Morton (1986) suggested, ecological, genetic, and physiological studies will also be necessary. If the genus Corbicula includes polyploid species that reproduce by gynogenesis, as our results indicate, it will be difficult to apply the biological species concept to these clams (Mayr and Ashlock, 199 1). The definition of species in Corbicula should be addressed after sufficient data on reproductive biology have been collected. Acknowledgments We especially thank Dr. W. L. Wu of the Institute of Zoology, Academica Sinica, Taipei, Taiwan, R.O.C., for providing valuable materials. We also thank Mr. T. Kawagishi of Mie University for technical assistance. This

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work was supported by a grant from the Science Technology Agency, Japan. Literature